1. Field of the Invention
The present invention relates to a method for producing L-glutamic acid by fermentation. L-Glutamic acid is widely used as a raw material for production of seasonings and so forth.
2. Description of the Related Art
L-glutamic acid is mainly produced by fermentation using an L-glutamic acid-producing bacterium of the so-called coryneform bacterium belonging to the genus Brevibacterium, Corynebacterium, Microbacterium, or mutant strains thereof. Moreover, methods utilizing a microorganism belonging to the genera Bacillus, Streptomyces, Penicillium, Pseudomonas, Arthrobacter, Serratia, Candida, Aerobacter aerogenes (currently Enterobacter aerogenes), a mutant strain of Escherichia coli, or the like, are known. Furthermore, also known are methods of producing L-glutamic acid using a microorganism belonging to the genera Klebsiella, Erwinia, or Pantoea (U.S. Pat. No. 6,197,559), and methods of producing L-glutamic acid using an Enterobacter bacterium (U.S. Pat. No. 6,331,419).
Furthermore, various techniques for improving L-glutamic acid-producing ability by enhancing activities of L-glutamic acid biosynthetic enzymes through the use of recombinant DNA techniques have been disclosed. For example, it was reported that introduction of a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective for enhancing L-glutamic acid-producing ability in bacterium belonging to the genus Corynebacterium or Brevibacterium (Japanese Patent Publication (Kokoku) No. 7-121228). In addition, Japanese Patent Laid-open (Kokai) No. 61-268185 discloses a cell harboring recombinant DNA containing a glutamate dehydrogenase gene derived from Corynebacterium bacteria. Furthermore, Japanese Patent Laid-open No. 63-214189 discloses a technique for increasing L-glutamic acid-producing ability by amplifying genes encoding glutamate dehydrogenase, isocitrate dehydrogenase, aconitate hydratase, and citrate synthase.
L-glutamic acid production has been considerably increased by the aforementioned breeding of microorganisms or the improving of production methods. However, in order to respond to an increased demand in the future, the development of methods which provide more efficient production of L-glutamic acid at a lower cost is still necessary, and therefore, still represent a need in the art.
Methods for L-glutamic acid fermentation while precipitating L-glutamic acid, which then accumulates in the culture broth, have been developed (European Patent Application Laid-open No. 1078989). Because the usual L-glutamic acid-producing bacteria cannot grow under acidic conditions, L-glutamic acid fermentation was conventionally performed under neutral conditions. Contrary to such conventional techniques, microorganisms which produce L-glutamic acid under acidic conditions were screened. As a result, it was reported that L-glutamic acid can be produced and accumulated in the medium while precipitating the L-glutamic acid by culturing the obtained microorganism (Enterobacter agglomerans) in a liquid medium in which pH was controlled so that L-glutamic acid is precipitated.
Furthermore, methods are known for producing L-glutamic acid by culturing such an L-glutamic acid-producing bacterium that can grow under acidic conditions, as described above, in a medium having a total content of organic acids that typically inhibit growth of the bacterium, but in an amount that does not inhibit the growth of the bacterium (European Patent Application Laid-open No. 1233070). Another known method includes a method for producing L-glutamic acid by culturing such a bacterium as described above at a first pH which is optimal for growth of the bacterium, and then culturing the bacterium at a second pH which is optimal for L-glutamic acid production by the bacterium, but is lower than the first pH (European Patent Application Laid-open No. 1233068).
Furthermore, a method is known for producing and accumulating L-glutamic acid in a medium while precipitating the L-glutamic acid into the medium, wherein crystals of L-glutamic acid are made to exist in the medium while the L-glutamic acid concentration in the medium is lower than the concentration at which natural crystallization of L-glutamic acid occurs (European Patent Application Laid-open No. 1233069).
The crystals of L-glutamic acid can exist in two forms, α- and β-form crystals (H. Takahashi, T. Takenishi, N. Nagashima, Bull. Chem. Soc. Japan, 35, 923 (1962); J. D. Bernal, Z. Krist., 78, 363 (1931); S. Hirokawa, Acta Cryst., 8, 637 (1955)). The β-form crystals are more stable in water, while the α-form crystals precipitate better, and are easier to handle in that, for example, the crystals can be effectively separated from a crystallized slurry. In the method described in European Patent Application Laid-open No. 1233069, the α-form crystals can be selectively crystallized when α-form crystals are used to induce the crystals of L-glutamic acid.
In addition, to lower the content of β-form crystals in L-glutamic acid crystals, methods of adding phenylalanine, leucine, tyrosine, cysteine, asparaginic acid, lysine, histidine, arginine, or alanine (Japanese Patent Publication (Kokoku) No. 36-17712, and Japanese Patent Publication (Kokoku) No. 38-16459), or adding ribonucleic acid, carboxy methyl cellulose, pectin, polyacrylic acid or salts thereof, or alginic acid or salts thereof (Japanese Patent Publication (Kokoku) No. 45-11286) have been disclosed. Those methods, however, have only been examined at the isoelectric point (pH 3.2) of L-glutamic acid, and no effect has been reported at a slightly higher pH, such as approximately pH 4.5.